2,2-Difluoroethyl Acetate

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2021-10-09 to 2022-11-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 92.9, 144, 223, 346, 537, 832, 1290 and 2000 mg/kg (nominal).
- Frequency of analytical investigation: concentrations were measured in the artificial soil at the beginning of the test (on day 0), on day 28 (when adults are removed) and at the end of the test (on day 56).
- Sample storage conditions before analysis: stored in refrigerator (2~8℃) after pretreatment if they were not analyzed directly within 24h.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil: approximately 29 g of test substance was dissolved in 1160 mL deionized water to prepare a 25 g/L stock solution. Then under cold storage conditions, appropriate volumes of this stock solution were quickly introduced into different small vials, with no headspace, intended to prepare the different test vessels. Then the stock solutions aliquoted in the separate vials were incorporated into formulated artificial soil to achieve the target concentrations. The treatment groups were prepared with a nominal moisture content of 40-60% by mass of the total water-holding capacity with deionized water. A mixer was used to blend the stock solution and the artificial soil. The details of each preparation of the test solutions are reported in “Any other information on materials and methods incl. tables” .
- Controls: The preparation process of the blank control group was the same as that of the test substance groups, but the test substance was not included.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworms
- Source: Jurong Breeding Farm, Jiangsu Province
- Weight at test initiation: 300 to 600 mg

ACCLIMATION
All newly acquired earthworms were quarantined and observed for 14 days prior to use in the test.
- Acclimation period and conditions: Earthworms were bred on a substrate consisting of 50% by volume peat and 50% by volume cow manure and kept under 20±2℃ for 7 days prior to testing. The test organisms with well developed clitellum were washed quickly before use and were transferred into artificial soil (without test substance) approximately 24 hours prior to the start of the test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Remarks:

The duration of the test was 8 weeks (56d). After the first 4 weeks (28d), the adult worms were removed. The next 4 weeks (28d) were dedicated to investigate effects on reproduction through the counting of newly hatched worms and cocoons.

Test temperature:

The measured temperature of the climate chamber remained between 20.8°C and 21.8°C throughout the study.

pH:

pH values varied from 6.00 to 6.18.

Moisture:

Soil moisture content varied from 23.13% to 27.22%.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1L breathable glass bottle with plastic cover
- Amount of soil or substrate: 500-600 g wet weight of the substrate
- No. of organisms per container (treatment): 10 earthworms
- No. of replicates per treatment group: 4
- No. of replicates per control: 8

SUBSTRATE
- Soil type: Artificial soil was used as a basic test substrate.
- Composition:
Sphagnum peat: 10% w/w
Kaolinite clay (Sigma-Aldrich): 20% w/w
Silica sand (screened with 0.2 mm sieve): 70% w/w
- pH: adjusted to 6.0±0.5.
- Maximum water holding capacity (WHC): Before the start of the test, the measured maximum WHC of the artificial soil was 48%. Moisture content of the test substrate was adjusted to 40-60% by mass of the total water-holding capacity with deionized water (Approximate moisture content is 19-29%).

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light and 8 hours dark
- Light intensity: 624 lux-670 lux
- Food: Food was first provided one day after adding the worms and applying the test chemical to the soil. 5 g of food were spread on the soil surface of each container and moistened with de-ionised water (5 ml per container). Thereafter food was provided once a week during the first 4-week test period. After removing the adults from the soil on day 28 of the test, a further 5 g of food then were administered to each test container. No further feeding took place during the remaining 4 weeks of the test.

EFFECT PARAMETERS MEASURED
- Toxicity parameters (definitive test):
* Mortality of adult worms on the 1st day and 28th day.
* Changes in behavior (e.g. inability to dig into the soil; lying motionless against the glass wall of the test vessel) and in morphology (e.g. open wounds) of surviving adult worms on the 28th day.
* Individual body weight of the adult worms on the 28th day.
* Numbers of newly hatched worms and cocoons on the 56th day.
* All signs of harm or damage to the newly hatched worms on the 56th day.
- Physico-chemical measurements: Test temperature and light intensity were recorded daily throughout the test with the exception of day-off. Soil moisture content and pH in the blank control and each test substance group were recorded at the start and termination of the test. Also, in order to check the soil humidity, the vessels were weighed at the beginning of the test (once containing the soil and the 10 earthworms) and thereafter at least once a week. Weight loss was replenished by the addition of an appropriate amount of deionised water. The water content should not vary by more than 10 % from that at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.55
- Range finding study
* Test concentrations: 0.18, 1.8, 18, 180 and 1800 mg/kg.
* Results used to determine the conditions for the definitive study: The mortality percentages were 0% after 14 days in the 0.18, 18, and 180 mg/kg treatment groups. The mortality percentage was 10% after 14 days in the 1.8 mg/kg treatment groups. The mortality percentage was 100% after 14 days in the 1800 mg/kg treatment group.

Nominal and measured concentrations:

- Nominal concentrations: 92.9, 144, 223, 346, 537, 832, 1290 and 2000 mg/kg.
- Measured concentrations:
* at T0: 37.2, 52.2, 105, 118, 206, 291, 405 and 786 mg/kg.
* on day1: * on days 28 and 56: The observation that the concentrations falled below the limit of detection over time is likely due the fact that the test substance is volatile.

Reference substance (positive control):

yes

Remarks:

Carbendazim

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

105 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LOEC

Effect conc.:

118 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC10

Effect conc.:

124 mg/kg soil dw

95% CI:

95 - 144

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

28 d

Dose descriptor:

LC50

Effect conc.:

179 mg/kg soil dw

95% CI:

157 - 198

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

52.2 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

LOEC

Effect conc.:

105 mg/kg soil dw

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

58.1 mg/kg soil dw

95% CI:

47 - 62.3

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

92.9 mg/kg soil dw

95% CI:

87.9 - 97.6

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

- Mortality at end of exposure period: The adult mortality percentage was 6.3% by the 28th day in the blank control group. In the 37.2, 52.2, 105, 118, 206, 291, 405 and 786mg/kg (initial measured concentrations) test substance groups, the adult mortality percentages on day 28 were 10, 7.5, 10, 22.5, 60, 100, 100 and 100%, respectively. According to the result of the Dunnett's Test, there was a statistically significant difference for 28 days mortality percentages between some test substance groups and the blank control group (P=1.0×10-25, P<0.05); the earthworms exposed to 118 mg/kg and to higher concentrations indeed showed significantly higher mortality percentages than blank control earthworms.

- Total mass of adults at beginning of test: See in "Any other information on results incl. tables".

- Changes in body weight of live adults after 28 days:
* Control: +2.64%
* 37.2 mg/kg (initial meas. conc.): +2.60%
* 52.2 mg/kg (initial meas. conc.): +2.85%
* 105 mg/kg (initial meas. conc.): +3.15%
* 118 mg/kg (initial meas. conc.): +3.52%
* 206 mg/kg (initial meas. conc.): +3.74%
* 291 mg/kg (initial meas. conc.): not measured due to 100% mortality at this concentration
* 405 mg/kg (initial meas. conc.): not measured due to 100% mortality at this concentration
* 786 mg/kg (initial meas. conc.): not measured due to 100% mortality at this concentration

- No. of offspring produced: The mean number of newly hatched worms per vessel at the end of the test (on day 56) in blank control group was 40, whereas this number was equal to 43, 40, 15, 15 and 2 in the 37.2, 52.2, 105, 118 and 206mg/kg test substance groups, respectively (all adult worms were dead at the highest concentrations of 291, 405 and 786mg/kg). According to the result of the Dunnett's Test, there was a statistically significant difference for 56 days reproductive output between some test substance groups and the blank control group (P=1.1×10-15, P<0.05). While the mean numbers of newly hatched worms in the test substances groups exposed to 37.2 and 52.2 mg/kg did not significantly differ from the blank control group, this number was significantly reduced in the test substances groups exposed to 105 (-62.5% as compared to control), 118 (-62.5%) and 206mg/kg (-95.0%).

- Morphological and behavioural abnormalities: No adverse behavioural effects and changes in appearance were observed after 28 days of exposure in adult earthworms in any of the test groups.

Results with reference substance (positive control):

Results with reference substance are valid. The mortality percentages in carbendazim treatment groups were shown to be between 0% to 85.0% and the reproduction outputs in carbendazim treatment groups were shown to be inhibited by 11.6 to 100% as compared to the solvent control group. The 56-days EC50 for Carbendazim was 0.430 mg/kg dry soil weight based on reproduction inhibition.

Reported statistics and error estimates:

Data were analyzed by appropriate statistical methods to calculate the LCx/ECX with 95% confidence limits and the NOEC/LOEC for reproduction and mortality using software Toxcalc v5.0.32.

Mortality of adult worms after 28 days of exposure:

Parameter	Blank control	Test substance groups (initial measured concentration, mg/kg dry soil weight)
		37.2	52.2	105	118	206	291	405	786
Duration (d)	28	28	28	28	28	28	1^§	1^§	1^§
Number of earthworms	80	40	40	40	40	40	40	40	40
Number of dead worms (A replicate)	1	1	1	1	2	7	10	10	10
Number of dead worms (B replicate)	1	1	0	1	3	5	10	10	10
Number of dead worms (C replicate)	0	1	1	1	1	5	10	10	10
Number of dead worms (D replicate)	0	1	1	1	3	7	10	10	10
Number of dead worms (E replicate)	0	—	—	—	—	—	—	—	—
Number of dead worms (F replicate)	1	—	—	—	—	—	—	—	—
Number of dead worms (G replicate)	1	—	—	—	—	—	—	—	—
Number of dead worms (H replicate)	1	—	—	—	—	—	—	—	—
Total	5	4	3	4	9	24	40	40	40
Mortality (%)	6.3	10.0	7.5	10.0	22.5*	60.0*	100*	100*	100*

^§ All worms were dead after one day in these test substance groups;

— Not applicable;

* Statistically significant difference as compared to the blank control group.

Morphology and behaviour of surviving adult worms after 28 days of exposure:

28d status description
Groups	Blank control	Test substance groups (initial measured concentration, mg/kg dry soil weight)
		37.2	52.2	105	118	206
	Number/ status	Number/ status	Number/ status	Number/ status	Number/ status	Number/ status
A	9/a	9/a	9/a	9/a	8/a	3/a
B	9/a	9/a	10/a	9/a	7/a	5/a
C	10/a	9/a	9/a	9/a	9/a	5/a
D	10/a	9/a	9/a	9/a	7/a	3/a
E	10/a	—	—	—	—	—
F	9/a	—	—	—	—	—
G	9/a	—	—	—	—	—
H	9/a	—	—	—	—	—

—: Not applicable;

a: normal, no special change.

 

Body weight of surviving adult worms after 28 days of exposure:

Groups			Number of worms day 0	Mean individual weight day 0 (mg)	Number of surviving worms day 28	Mean individual weight of surviving worms day 28 (mg)	Mean individual weight reduction after 28 days ±standard deviation (%)
Blank control		A	10	356	9	353	2.64±1.51
		B	10	348	9	343
		C	10	349	10	340
		D	10	351	10	347
		E	10	362	10	351
		F	10	373	9	361
		G	10	348	9	329
		H	10	356	9	344
Test substance groups (measured concentration, mg/kg dry soil weight)	37.2	A	10	358	9	350	2.60±0.63
		B	10	341	9	334
		C	10	339	9	330
		D	10	346	9	334
	52.2	A	10	353	9	342	2.85±0.29
		B	10	357	10	346
		C	10	341	9	332
		D	10	351	9	342
	105	A	10	360	9	347	3.15±0.77
		B	10	341	9	332
		C	10	338	9	330
		D	10	352	9	338
	118	A	10	356	8	344	3.52±0.25
		B	10	369	7	357
		C	10	355	9	342
		D	10	343	7	330
	206	A	10	355	3	337	3.74±1.04
		B	10	350	5	338
		C	10	362	5	348
		D	10	349	3	340

 

Number of newly hatched worms after 56 days:

Parameter	Blank control	Test substance groups (initial measured concentration, mg/kg dry soil weight)
		37.2	52.2	105	118	206
Duration (d)	56	56	56	56	56	56
Number of newly hatched worms (A replicate)	40	42	39	14	13	3
Number of newly hatched worms (B replicate)	38	47	41	17	15	1
Number of newly hatched worms (C replicate)	32	43	36	12	14	1
Number of newly hatched worms (D replicate)	41	39	44	15	19	2
Number of newly hatched worms (E replicate)	43	—	—	—	—	—
Number of newly hatched worms (F replicate)	44	—	—	—	—	—
Number of newly hatched worms (G replicate)	44	—	—	—	—	—
Number of newly hatched worms (H replicate)	37	—	—	—	—	—
Mean	40	43	40	15	15	2
CV (%)	10.0	6.98	7.50	13.3	20.0	50.0
Inhibition of the reproductive output as compared to blank control (%)^§	—	[7.50]	0	62.5*	62.5*	95.0*

—: Not applicable;

§ : A value between brackets does not refer to an inhibition of the reproductive output, but to the fact that the number of newly hatched worms produced was higher as compared to the blank control group;

*: Statistically significant difference as compared to the blank control group.

Validity criteria fulfilled:

yes

Remarks:

Mortality was 6.3% by the 28th day in the blank control group (< 10%). Mean number of newly hatched worms produced by 10 adults per vessel at the end of the test in blank control group was 40 (> 30) with a coefficient of variation of 10.0% (< 30%).

Conclusions:

The 56 days EC10 of 2,2-Difluoroethyl acetate to Eisenia fetida based on reproduction was determined to be 58.1 mg/kg dry soil.

Executive summary:

The chronic toxicity of 2,2-Difluoroethyl acetate to Eisenia fetida was investigated in a 56-days test according to the OECD guideline 222 under GLP compliance.

Groups of 40 earthworms (divided in 4 replicates of 10 earthworms each) were exposed to test chemical at nominal concentrations of 92.9, 144, 223, 346, 537, 832, 1290 and 2000 mg/kg (dry artificial soil weight). In parallel, 80 earthworms (divided in 8 replicates of 10 earthworms each) were exposed to a control treatment. Gas Chromatography with Flame Ionisation Detection (GC-FID) analysis showed that at the beginning of the test, the initial measured concentrations of the test substance in the artificial soils were 37.2, 52.2, 105, 118, 206, 291, 405 and 786 mg/kg, respectively. On day 1, the measured concentrations of test substance in the groups nominally exposed to 92.9 to 1290 mg/kg were lower than the the limit of detection of the analytical method (i.e. <LOD, LOD=2.54mg/kg), while the mean measured concentration of test substance in the group nominally exposed to 2000 mg/kg was 5.80mg/kg. It has to be noticed that on day 1, all the adult worms in the treatment groups exposed to the three highest concentrations were dead, meaning these effects were related to the initial measured concentration of test substance in artificial soils. On day 28 (when the adult worms were removed) and at the end of the test on day 56, the measured concentrations in the artificial soil for all test substance groups were lower than LOD (i.e. <2.54 mg/kg). The observation that concentrations falled below the limit of detection over time was likely due the fact that the test substance is volatile. Since effects were observed during this test and these effects were related to the initial measured concentration of test substance in artificial soils, all test results were expressed based on these initial measured concentrations.

On day 28, the adult mortality percentages were 6.3, 10, 7.5, 10, 22.5, 60, 100, 100 and 100% in the blank control and 37.2, 52.2, 105, 118, 206, 291, 405 and 786 mg/kg (initial measured concentrations) test substance groups, respectively. Under the tested conditions, the 28 days LC10 for mortality of 2,2-Difluoroethyl acetate to adult earthworms was 124 mg/kg (95% confidence limit was 95.0-144 mg/kg).

On day 56, the numbers of newly hatched worms in the test substance groups exposed to 37.2 and 52.2 mg/kg did not significantly differ from the blank control group, but these numbers were reduced by 62.5, 62.5 and 95.0% in the test substances groups exposed to 105, 118 and 206 mg/kg (initial measured concentrations). Under the tested conditions, the 56 day EC10 for reproduction of 2,2-Difluoroethyl acetate to Eisenia fetida was determined to be 58.1 mg/kg (95% confidence limit was 47.0-62.3 mg/kg).

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Fom 2021-10-09 to 2022-11-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Version / remarks:

1984

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 164, 270, 445, 735, 1212 and 2000 mg/kg (nominal).
- Frequency of analytical investigation: concentrations were measured in the artificial soil at the beginning of the test, on days 7 and 14 in each replicate of the control and each test substance group.
- Sample storage conditions before analysis: stored in refrigerator (2~8℃) after pretreatment if they were not analyzed directly within 24h.

Vehicle:

no

Details on preparation and application of test substrate:

- Method of mixing into soil: approximately 29 g of test substance was dissolved in 1160 mL deionized water to prepare a 25 g/L stock solution. Then for each test, appropriate volumes of this stock solution were incorporated into formulated artificial soil to achieve the target concentrations. The treatment groups were prepared with a nominal moisture content of 40-60% by mass of the total water-holding capacity with deionized water. A mixer was used to blend the stock solution and the artificial soil. The details of each preparation of the test solutions are reported in “Any other information on materials and methods incl. tables"
- Controls: The preparation process of the blank control group was the same as that of the test substance groups, but the test substance was not included.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
- Common name: earthworms
- Source: Jurong Breeding Farm, Jiangsu Province
- Weight at test initiation: 300 to 600 mg
- Feeding during the definitive test: No

ACCLIMATION
All newly acquired earthworms were quarantined and observed for 14 days prior to use in the test.
- Acclimation period and conditions: Earthworms were bred on a substrate consisting of 50% by volume peat and 50% by volume cow manure and kept under 20 ± 2℃ for 7 days prior to testing. The test organisms with well developed clitellum were washed quickly before use and were transferred into artificial soil (without test substance) approximately 24 hours prior to the start of the test.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

19.7 °C to 20.3 °C

pH:

6.28 to 6.42

Moisture:

31.96% to 34.07%

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 1 L breathable glass bottle with plastic cover
- Amount of soil or substrate: 750 g wet weight of the substrate
- No. of organisms per container (treatment): 10 earthworms
- No. of replicates per treatment group: 4
- No. of replicates per control: 4

SUBSTRATE
- Soil type: Artificial soil was used as a basic test substrate.
- Composition:
Sphagnum peat: 10% w/w
Kaolinite clay (Sigma-Aldrich): 20% w/w
Silica sand (screened with 0.2 mm sieve): 70% w/w
- pH: adjusted to 6.0±0.5.
- Maximum water holding capacity (WHC): Before the start of the test, the measured maximum WHC of the artificial soil was 70%. Moisture content of the test substrate was adjusted to 40-60% by mass of the total water-holding capacity with deionized water (Approximate moisture content is 28-42%).

OTHER TEST CONDITIONS
- Photoperiod: Continuous illumination.
- Light intensity: between 630-710 lux.

EFFECT PARAMETERS MEASURED
- Toxicity parameters: Mortality on the 7th and 14th day. Changes in behaviour (e.g. inability to dig into the soil; lying motionless against the glass wall of the test vessel) and in morphology (e.g. open wounds) were also recorded on the 7th and 14th day.
- Physico-chemical measurements: During the test, the test temperature was continuously monitored and the illumination was recorded every working day. At the beginning and the end of the test, the soil moisture content and pH value of one test container were recorded in the blank control group and the test substance groups randomly.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.65
- Range finding study
* Test concentrations: 0.18, 1.8, 18, 180 and 1800 mg/kg
* Results used to determine the conditions for the definitive study: The mortality percentages were 0% after 14 days in the control, 0.18, 18, and 180 mg/kg treatment groups. The mortality percentage was 10% after 14 days in the 1.8 mg/kg treatment group. The mortality percentage was 100% after 14 days in the 1800 mg/kg treatment group.

Nominal and measured concentrations:

- Nominal concentrations: 164, 270, 445, 735, 1212 and 2000 mg/kg
- Measured concentrations:
* at T0: 58.2, 113, 162, 283, 456 and 740 mg/kg
* On days 7 and 14: the measured concentrations for all treatment groups were lower than the detection limit (LOD was 2.54 mg/kg).

Reference substance (positive control):

yes

Remarks:

chloroacetamide

Duration:

7 d

Dose descriptor:

LC50

Effect conc.:

202 mg/kg soil dw

95% CI:

181 - 224

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

193 mg/kg soil dw

95% CI:

146 - 256

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mortality

Details on results:

- Mortality at end of exposure period: No mortality occurred in the blank control group as observed during the assessments on the 7th and 14th day. The mortality percentages were 0% after 7 days and 2.5% after 14 days in the 58.2 mg/kg treatment group, and 15% after 7 days and 20% after 14 days in the 113 mg/kg treatment group. Mortality percentages in the 162 mg/kg treatment group after 7 and 14 days were both 20%, and were both 77.5% in the 283 mg/kg treatment group. Mortality percentages in the 456 and 740 mg/kg treatment groups after 7 and 14 days were 100%.
- Total mass of adults at beginning of test: as mean individual weight:
* Control: 359 to 395 mg
* 58.2 mg/kg (initial meas. conc.): 366 to 413 mg
* 113 mg/kg (initial meas. conc.): 377 to 406 mg
* 162 mg/kg (initial meas. conc.): 374 to 393 mg
* 283 mg/kg (initial meas. conc.): 377 to 413 mg
* 456 mg/kg (initial meas. conc.): 363 to 394 mg
* 740 mg/kg (initial meas. conc.): 359 to 373 mg
- Changes in body weight of live adults at end of exposure period:
* Control: -11.04%
* 58.2 mg/kg (initial meas. conc.): -9.07%
* 113 mg/kg (initial meas. conc.): -8.72%
* 162 mg/kg (initial meas. conc.): -14.43%
* 283 mg/kg (initial meas. conc.): -12.59%
* 456 mg/kg (initial meas. conc.): not measured due to 100% mortality at this concentration
* 740 mg/kg (initial meas. conc.): not measured due to 100% mortality at this concentration
- Morphological and behavioural abnormalities: No adverse behavioural effects and changes in appearance were observed after either 7- or 14-day exposure in any of the treatment groups.

Results with reference substance (positive control):

Results with reference substance are valid. The 14-days LC50 for chloroacetamide was 20.1 mg/kg dry soil weight (historical range of the testing lab: 13 to 25.6 mg/kg).

Reported statistics and error estimates:

Data were analyzed to calculate the LC50 with 95% confidence limits on 7th and 14th day using software Toxcalc v5.0.32.

The cumulative mortality resulting from the exposure of Eisenia fetida to the test substance during the definitive test is given below:

Parameter	Blank control group		Test substance groups (Initial measured concentration, mg/kg)
			58.2		113		162		283		456		740
Duration (d)	7	14	7	14	7	14	7	14	7	14	7	14	7	14
Number of earthworm	40	40	40	40	40	40	40	40	40	40	40	40	40	40
Number of dead earthworms (A replicate)	0	0	0	1	1	3	0	0	6	6	10	10	10	10
Number of dead earthworms (B replicate)	0	0	0	0	1	1	0	0	10	10	10	10	10	10
Number of dead earthworms (C replicate)	0	0	0	0	1	1	4	4	10	10	10	10	10	10
Number of dead earthworms (D replicate)	0	0	0	0	3	3	4	4	5	5	10	10	10	10
Total mortality	0	0	0	1	6	8	8	8	31	31	40	40	40	40
Mortality (%)	0	0	0	2.5	15	20	20	20	77.5	77.5	100	100	100	100

Since acute toxicity effects were observed during this test and these effects were related to the initial measured concentration of test substance in artificial soils, all test results were expressed based on these initial measured concentrations.

Validity criteria fulfilled:

yes

Remarks:

The mortality was 0% in the blank control group.

Conclusions:

The 14 day LC50 of 2,2-Difluoroethyl acetate to Eisenia fetida was determined to be 193 mg/kg dry soil.

Executive summary:

The acute toxicity of 2,2-Difluoroethyl acetate to Eisenia fetida was investigated in a 14-days test according to the OECD guideline 207 under GLP compliance.

Groups of 40 earthworms (divided in 4 replicates of 10 earthworms each) were exposed to control, and test chemical at nominal concentrations of 164, 270, 445, 735, 1212 and 2000 mg/kg (dry artificial soil weight). Gas Chromatography with Flame Ionisation Detection (GC-FID) analysis showed that at the beginning of the test, the initial measured concentrations of the test substance in the artificial soils were 58.2, 113, 162, 283, 456 and 740 mg/kg, respectively. The measured concentrations of the test substance in the artificial soils after both 7 and 14 days were lower than the limit of detection of the analytical method (i.e. <2.54 mg/kg). Since acute toxicity effects were observed during this test and these effects were related to the initial measured concentration of test substance in artificial soils, all test results were expressed based on these initial measured concentrations.

No mortality occurred in the blank control group as observed during the assessments on the 7th and 14th day. The mortality percentages were 0% after 7 days and 2.5% after 14 days in the 58.2 mg/kg treatment group, and 15% after 7 days and 20% after 14 days in the 113 mg/kg treatment group. Mortality percentages in the 162 mg/kg treatment group after 7 and 14 days were both 20%, and were both 77.5% in the 283 mg/kg treatment group. Mortality percentages in the 456 and 740 mg/kg treatment groups after 7 and 14 days were 100%.

Under the conditions of the test, the 14-Day LC50 for the test substance to earthworms (Eisenia fetida) was 193 mg/kg dw and the 95% confidence interval was from 146 to 256 mg/kg.

Description of key information

Short-term toxicity to earthowrms: The 14 day LC50 of 2,2-Difluoroethyl acetate to Eisenia fetida was determined to be 193 mg/kg dry soil.

Long-term toxicity to earthowrms: The 56 day EC10 of 2,2-Difluoroethyl acetate to Eisenia fetida was determined to be 58.1mg/kg dry soil based on reproduction inhibition.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

193 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

58.1 mg/kg soil dw

Additional information

Two experimental studies performed under GLP compliance and in accordance with OECD test guidelines 207 and 222 were assigned a Klimisch score of 1 and flagged as key studies for short-term and long-term toxicity, respectively. The acute toxicity test reports a 14-day LC50 of 193 mg/kg dry soil in Eisenia fetida. The chronic toxicity test reports a 56-day EC10 for reproduction of 58.1 mg/kg dry soil in the same species.